- Volume 68, Issue 3, 1987

A radioimmunoprecipitation assay (RIPA) was used to study the serum antibody responses to individual polypeptides that developed after infection with viruses from human rotavirus subgroups I and II. Paired sera from eight children (1 to 8·5 years of age) were used in the study. Although all of the eight acute sera were negative by the complement fixation test, four of them were positive by RIPA, indicating a previous infection by rotavirus. A significant difference in the number of polypeptides immunoprecipitated was seen among the convalescent sera. The number of polypeptides immunoprecipitated was found to be related to previous infection experience. At most, ten different polypeptides were immunoprecipitated: seven structural polypeptides VP1 to VP7 and three non-structural polypeptides, NS1, NS2 and NS3. No sera immunoprecipitated VP8 or VP9. Acute sera positive by RIPA immunoprecipitated up to five polypeptides, VP1, VP2, VP3, VP4 and VP6. One of the non-structural proteins (NS2) was found to be particularly immunogenic, since antibodies to this polypeptide were detected in several convalescent sera. Among the structural proteins VP2 and VP6 were found to be the two immunodominant polypeptides which were recognized by all convalescent sera. Only three convalescent sera immunoprecipitated VP7, the major type-specific antigen responsible for inducing neutralizing antibodies. Three of four originally seronegative children with no reactivity in the convalescent sera to VP7 developed neutralizing antibodies to a single serotype. One child developed antibodies to two serotypes.

Analysis of the genomic dsRNA of rotaviruses isolated from calves with subclinical infections has revealed eight calves excreting group A viruses with unusual genome profiles. Two of these virus strains, C7–176 and C7–183, were grown and cloned by plaque purification in cell culture. Examination of their genome profiles after cloning showed that the unusual pattern had been retained. They were without RNA segment 11 but had an extra band (6a) migrating between segments 6 and 7. However, they contained the triplet of segments 7, 8 and 9, of similar size, which is characteristic of group A rotaviruses. The number and mol. wt. of the intracellular polypeptides induced by these viruses were similar to those of the bovine group A rotavirus UK strain. Analysis of the RNA transcripts produced by the transcription in vitro of purified C7–183 virus showed that segment 6a produced a large RNA transcript of corresponding size. After isolation, this transcript was translated in a rabbit reticulocyte lysate preparation and yielded a single polypeptide, vpll, equivalent to the product of segment 11 of rotaviruses with typical genome profiles.

Eight viruses of the Corriparta serogroup (Reoviridae: Orbivirus) that were known to be heterogeneous on the basis of serology and polyacrylamide gel electrophoresis were examined by reciprocal RNA-RNA blot hybridization of genomic RNA. Conserved and variant genes were identified by the degree of hybridization between cognate genes of different isolates. The eight viruses were divided into three subsets on the basis of the number of shared genes. Four of the viruses, isolated in Australia, formed one subset of related isolates and shared five conserved genes. Another isolate, Acado, was variant in all 10 genes and was considered to be a second subset. The remaining three isolates formed a third subset and shared four conserved genes. Genes 1, 3 and 10 were the most variable among the Corriparta serogroup isolates. Subsets of isolates within a serogroup which are highly related in the majority of the 10 genes and less related to serogroup viruses in another subset have not been reported previously. The phylogenetic relationship of Corriparta serogroup members suggested by the blot hybridization data is not apparent in the current taxonomic classification of these viruses which is based primarily upon serological data. The hybridization data on the Corriparta serogroup viruses are discussed and contrasted with other Orbivirus serogroups which have been examined similarly.

DNA from Molluscum contagiosum virus (MCV) isolates was analysed by restriction endonuclease cleavage, revealing two virus subtypes. Physical maps of cleavage sites for BamHI, ClaI and HindIII were constructed, and found to differ extensively between the two subtypes. MCV DNA was similar to Orthopoxvirus DNA with respect to size, terminal cross-linking and the presence of inverted terminal repetitions, but did not hybridize with vaccinia virus DNA. The genomes of the two MCV subtypes cross-hybridized and were colinear except for two small regions. There was sequence homology between DNA from corresponding map regions of the MCV subtypes but, in contrast to Orthopoxvirus DNA, no conservation of restriction sites. A synthetic oligonucleotide probe representing a conserved domain of epidermal growth factor, μ-transforming growth factor and the vaccinia growth factor identified equivalent regions of both MCV genomes as having the potential to encode this domain. This locus is similar to the position of the vaccinia growth factor gene in vaccinia virus DNA. Thus MCV may induce epidermal cell proliferation and tumourigenesis by expression of an epidermal growth factor-like polypeptide.

A 12·4 kbp HindIII chromosomal DNA fragment harbouring an apparently intact 9·2 kbp endogenous murine leukaemia virus (MuLV)-related proviral genome was isolated from an RFM/Un strain mouse by molecular cloning and designated pRFM # 6. Nucleotide sequence analysis revealed the following characteristic features in the pRFM # 6 provirus: a distinct 200 bp sequence in the long terminal repeat (LTR) mid-U3 region, a primer binding site for glutamine tRNA, a 3′ pol region encoding an ‘endonuclease’ protein of 390 amino acids, and the mink cell focusforming virus type-specific sequence at the 5′ portion of the env gene. The 699 bp 5′ LTR and 700 bp 3′ LTR of pRFM # 6 provirus were identical except for three base changes in the U3 ‘enhancer’ region. At the cell-provirus DNA junction, 4 bp direct repeats were present. The proviral genome was found at the same chromosomal DNA site in BALB/c, AKR, C3H, CBA and RFM strain mice, but not in NFS/N or C57BL/6 strain mice.

Incubation period and survival time were determined in C57BL mice which had been injected stereotaxically with either the 139A, ME7 or 22L strain of scrapie in one of five different brain regions (cerebral cortex, caudate nucleus, thalamus, substantia nigra, cerebellum). The injection of 139A in the caudate nucleus, thalamus, substantia nigra or cerebellum resulted in significantly shorter incubation periods than following cerebral cortex injection. For ME7, mice injected in the thalamus and cerebellum had incubation periods approximately 2 weeks shorter than the cerebral cortex group. For 22L, the incubation periods after substantia nigra or cerebellum injection were significantly shorter than after cortex injection. The cerebellum injection group had a significantly shorter incubation period than the substantia nigra injection group. The survival times for mice injected with a particular scrapie strain were directly related to the incubation periods. The groups with the shortest incubation also had the shortest survival time (e.g. 22L in the cerebellum). On histological examination, 139A and ME7 produced brain lesions in all brain areas regardless of injection site. For the 22L strain, after cerebellum injection vacuolation was limited to the cerebellum, while injection into the cerebral cortex and other forebrain regions resulted in vacuolation in all brain regions examined. Despite the difference in the distribution of vacuolation seen in cerebellum- compared to cortex-injected (22L) mice, infectivity titres were similar in the cortex, cerebellum, cerebellar cortex and medulla plus pons.

The neurovirulence of eight temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 in 4-week-old BALB/c mice was investigated. Whereas a dose of 100 p.f.u. of wild-type virus killed mice within a week, a 1000-fold higher dose of ts mutants did not. Three ts mutants induced demyelinating disease in the central nervous system (CNS). The pathology of the demyelinating disease caused by one mutant, designated ts-342, was studied in detail. Pathological changes, starting 3 days post-inoculation (p.i.), were characterized by inflammation and demyelination in the CNS. Antibody responses directed against all virus-specific structural proteins were present at 7 days p.i. No virus particles were observed by electron microscopy at 14 days p.i. However, macrophages and lymphocytes were abundant in the areas of demyelination. The growth kinetics in vivo of wild-type virus, ts-342 and a revertant of ts-342 were compared. Wild-type virus and the revertant replicated rapidly in the brain and spread to the liver causing a lethal hepatitis. Ts-342, however, replicated to a much lesser extent within the brain and could not be detected in the blood or liver. The ts lesion in the genome of ts-342 seems, therefore, to determine the outcome of the infection.

Summary: Spikes of different kinds, distinct in size and appearance were detected on the surfaces of herpes simplex virions by electron microscopy of negatively stained preparations. Use of monoclonal antibodies coupled to colloidal gold permitted identification of viral glycoproteins present in different structures projecting from the virion envelope. Antibodies specific for the glycoprotein designated gB bound to the most prominent spikes, which were about 14 nm long and, in side view, had a flattened T-shaped top. Antibodies specific for gC bound to structures that, in some instances, appeared to extend as much as 24 nm from the surface of the envelope and were too thin to resolve. Antibodies specific for gD bound to structures that extended as much as 8 to 10 nm from the surface of the envelope. The gB spikes were invariably clustered, usually in protrusions of the envelope varying from small bulbous distentions to long tail-like projections. The gC components were randomly distributed and widely spaced and the gD components were irregularly clustered in patterns distinct from those of the gB spikes. These three glycoproteins therefore form structures that are different in size, morphology and distribution in the envelope.

The nuclear matrix is involved in the replicative cycle of herpes simplex virus type 1 (HSV-1) and in at least some cases viral DNA has been shown to be closely associated with this structure. In this communication, we report the presence of five DNA- binding proteins in the nuclear matrix of HSV-1-infected BHK cells. These proteins (p114, p89, p77, p37 and p29) were detected by probing with 32P-labelled HSV DNA after Western blotting of nuclear matrix proteins. Three were identified as virion components: p89 as VP12, p77 as VP13 and p37 as the capsid protein VP22a. These polypeptides were detected in cells and nuclei and found to be associated with the nuclear matrix late during the lytic cycle, long after the onset of viral DNA replication.

The nuclear matrix-binding capacity of VP22a depended on viral DNA replication, since after DNA polymerase inhibition it was still synthesized and transported into the nucleus but was no longer associated with the nuclear matrix. After inhibition of viral DNA synthesis, VP13 was no longer found in cells, nuclei or nuclear matrices. These results suggest a possible involvement in anchoring viral progeny DNA to the nuclear matrix.

Herpes simplex virus type 1 (HSV-1) infection of human fibroblast cells grown in culture induces reorganization of the cytoskeleton fibrillar structures. Normal transport and insertion of HSV glycoproteins into the plasma membrane of the cells depend on the integrity of the microtubules. The natural host cells for HSV are epithelial cells, and an epithelial cell line established from rat palate was used in the present study. The effect of virus on the structure of the intermediate filaments and especially on the keratin proteins was studied. Two-dimensional gel electrophoresis of total cell extracts identified in uninfected cells two major acidic keratin proteins with apparent molecular weights of 44000 (44K) and 48K (pI 5·45 to 5·30, 5·50 to 5·35). A new keratin protein of 46K (pI 5·40 to 5·25) appeared in infected cells between 8 h and 12 h post-infection. Pulse-chase experiments identified the 46K protein as a processed form of the 48K keratin component, which was also cleaved in uninfected cells grown in the presence of cycloheximide. Partial proteolysis of the 46K and 48K keratins with Staphylococcus aureus V8 protease showed that the 48K and the 46K proteins differed in only one oligopeptide. The significance of the changed keratin composition of HSV- infected cells is discussed.

One-hundred and seventy-two epidemiologically unrelated herpes simplex virus type 1 (HSV-1) strains isolated in Japan (104 strains) and Sweden (68 strains) were compared by analysis of their genome structures using four restriction endonucleases, BamHI, KpnI, SalI and HindIII In addition, 32 Kenyan HSV-1 isolates previously compared to Japanese isolates were included for further comparison with the Swedish isolates. Remarkable and statistically significant differences were found between the HSV-1 isolates from the three countries. One-hundred and thirty cleavage sites were examined, and it was shown that isolates from two of the three countries were statistically distinguishable at 27 of these loci. Pairwise comparison between isolates from Japan and Sweden, Kenya and Sweden, and Japan and Kenya revealed variation in 18, 16 and 23 sites, respectively. By considering gains and losses of 19 sites, the total of 204 strains could be classified into 92 distinct cleavage patterns. Isolates from the three countries could be distinguished from each other by the pattern, except for one Swedish and two Kenyan isolates which shared a pattern. Twenty-one fragments that were present or absent only in individual isolates from one or other of the three countries could be detected. These results show that HSV-1 strains from geographically separate countries or anthropologically different races have distinct distributions of endonuclease recognition sites.

A HindIII cleavage map of the genome DNA of a new isolate of human cytomegalovirus (HCMV), strain Tanaka, was constructed by cosmid cloning and Southern blot hybridization of virion DNA. The genome was found to be unique in that its long (L) component was composed of two subsegments, L1 and L2, and subsegment L2 underwent inversion relative to L1 at high frequency. In addition to the normal inversions of the L and short (S) components, this produced eight genome isomers. The novel invertible subsegment was flanked by an inverted sequence distinct from the inversion-specific a sequence present in the terminal and junction regions of the genome.

We previously reported that a host protein, β 2 microglobulin (β 2 m) inhibited the detection of human cytomegalovirus (CMV) in urine specimens by enzyme immunoassay and postulated that β 2 m bound to the virus particle and masked the viral antigenic determinants. We report here that CMV strain AD169 grown in cell culture bound human β 2 m when this protein was added to cell culture fluids or when the virus was added to urine. Such binding was not seen with herpes simplex virus. CMV could also bind bovine β 2 m from foet al calf serum in cell culture fluids. The use of radiolabelled β 2 m in other experiments showed that CMV bound β 2 m after release from cells and that the bound β 2 m did not represent acquisition of class I HLA molecules during budding from host cell membranes. Immunoprecipitation studies showed that β 2 m was bound by two viral envelope proteins β 2 m BP1 (β 2 m-binding protein 1) and β 2 m BP2 of molecular masses 36000 and 65000 daltons respectively. β 2 m could not bind to separated viral proteins under reducing or non-reducing conditions. We propose that interaction of these two proteins on the viral surface is required to enable CMV to bind β 2 m.

We have previously reported that human cytomegalovirus (CMV) from urine specimens cannot be captured onto a solid phase by CMV-specific monoclonal antibodies that can capture CMV grown in vitro. We report here that CMV exists in vivo in body fluids such as urine as β 2 microglobulin (β 2m)-coated particles. We have demonstrated the presence of β 2m on CMV purified directly from urine by Western blotting and have shown that the β 2m was associated with the viral envelope. Urinary CMV could be specifically bound by an affinity column comprising a monoclonal antibody specific for β 2m bound to Sepharose. The β 2m-coated urinary CMV could not be neutralized by hyperimmune globulin, human immune sera or murine monoclonal antibodies that could neutralize CMV grown in cell culture. We conclude that the binding of β 2m by CMV masks the important antigenic sites necessary for neutralization which are recognized by man’s immune response. We propose that CMV has evolved this mechanism of coating itself in a host protein as a mechanism of evading the host immune response and facilitating transmission between individuals.

SUMMARY We have previously demonstrated that human cytomegalovirus (CMV) binds the host protein β 2 microglobulin (β 2m) from body fluids or from cell culture media. In this report we have examined the effect of the β 2m on viral infectivity. We have shown that the addition of human purified β 2m, or a fraction of foetal calf serum corresponding to bovine β 2m, to culture medium increased the amount of infectious extracellular CMV, compared to that from cells grown in serum-free medium. Metabolic labelling experiments demonstrated that this effect was not due to an increase in the amount of extracellular virus but to an increase in the infectivity of the virus present in extracellular fluids. We concluded that the binding of β 2m by CMV increased its infectivity. We have shown that CMV and β 2m compete for binding sites on fibroblasts. As the main binding site on cells for /Cm is the class IHLA heavy chain we compared the binding of CMV to the Raji and Daudi cell lines which express or lack expression of class I HLA molecules. The binding of radiolabelled βm-coated CMV was significantly higher to Raji cells than to Daudi cells. Furthermore, CMV could compete with β 2m for binding to Raji cells, although the reverse was not true. These results demonstrate that CMV can use class I HLA molecules as a virus receptor. We propose that when coated with β 2Cm, CMV has the capacity to displace β 2m from the class I HLA heavy chain-βm dimer on the cell surface and bind to cells. The fact that β 2m enhances infectivity suggests that such binding leads to productive infection of cells.

Cultured human epithelium bound and internalized radiolabelled Epstein-Barr virus (EBV) within 1 h of exposure. A similar percentage of cultured cells also were reactive with monoclonal antibodies to the EBV/C3d receptor of B lymphocytes. In cross-sections of fresh frozen, stratified epithelium, receptor expression seemed limited to the less differentiated subpopulation of cells. These findings support the notion of direct infection of epithelial cells by EBV and suggest a viral life cycle in epithelium dependent on the stage of cell differentiation.

Herpes simplex virus type 1 (HSV-1) has been isolated from explanted human corneas after cultivation in vitro. To determine whether HSV-1 is persistent or latent in corneal cells, a system to study HSV-1 infection of rabbit corneal cells in vitro was developed. By elevation of the incubation temperature to 42 °C before and during HSV-1infection it was shown that both keratocytes and epithelial cells support a nonproductive rather than a productive infection. On subsequent temperature reduction to 37 °C, infectious virus was released from both cell types. Addition of the viral inhibitor acycloguanosine during the last 5 days of a 14 day incubation at 42 °C did not reduce the frequency of viral shedding following transfer to 37 °C, indicating that corneal cells support a latent as opposed to persistent infection. Cultures which failed to shed virus spontaneously up to 29 days post-inoculation were superinfected at 37 °C, with an Xba\ site deletion mutant of HSV-1. Restriction endonuclease analysis of progeny identified both the initial infecting virus and recombinants between the parental and superinfecting genomes.

Rat monoclonal antibodies were used to deplete selectively Lyt 2 (cytotoxic) and L3T4 (helper) T cell populations in vivo. These antibodies produced >95 % depletion of the respective T cell subset as determined by fluorescent antibody and cytofluoro-graphic analyses. Antibody-treated mice were infected in the ear pinna with herpes simplex virus (HSV) and the induction of virus-specific T cell and antibody responses were monitored during the acute infection. Lyt 2-deficient mice produced delayed hypersensitivity and HSV-specific antibodies comparable to those in untreated animals. However, major histocompatibility complex class I-restricted T cell killing was abolished. In contrast, L3T4-deficient animals failed to produce either primary delayed hypersensitivity response or specific antibodies to the virus, but cytotoxic T cell responses were induced and even augmented in comparison with infected, normal animals. This observation clearly demonstrates that Lyt 2 cytotoxic T cells can be induced in a helper T cell-deficient environment. The ability of T cell subset-deficient mice to clear infectious virus was investigated in the skin of the ear and the part of the nervous system innervating the site of infection. L3T4-deficient animals showed a markedly delayed clearance of virus from the ear and also had a more florid infection of the nervous system. However, Lyt 2-deficient mice cleared the infection in the ear normally, but a severe infection of the nervous system was still observed. The implication of these observations to the pathogenesis of this virus is discussed.